Letter to the Editor
Detection and characterization of OXA-48-producing Klebsiella pneumoniae originated in Bulgaria Stefana Sabtcheva1, Ivan N. Ivanov2, Bozhana Todorova2, Yordan Simeonov3, Elina Dobreva2, Krasimira Ivanova2, Tzvetan Velinov2, Todor Kantardjiev2 1
Laboratory for Clinical Microbiology, National Oncology Center – SHATO, Sofia, Bulgaria, 2National Reference Laboratory for Control and Monitoring of Antibiotic Resistance, Department of Microbiology, National Center of Infectious and Parasitic Diseases, Bulgaria, 3Department of Surgery, Specialized Hospital for Active Treatment in Oncology – SHATO, Sofia, Bulgaria
We report the identification of OXA-48-producing Klebsiella pneumoniae, causing peritonitis in a cancer patient admitted to the Oncology Hospital in Sofia. The isolate had reduced susceptibility to carbapenems but remained susceptible to extended-spectrum cephalosporins. PCR and sequencing confirmed the presence of blaOXA-48 gene flanked by two intact copies of IS1999 on truncated DTn1999.1. This transposon was located on unusual non-typeable 29-kb plasmid that could be transferred only by transformation. Multilocus sequence typing (MLST) indicated the presence of the sequence type ST530.This is the first documented infection due to OXA-48-producing Enterobacteriaceae strain in Bulgaria. Keywords: Carbapenem resistance, Oxacillinases, OXA-48, Klebsiella pneumoniae
Introduction Carbapenemase-mediated resistance in Enterobacteriaceae has increased in the last years, seriously compromising the management of life-threatening infections. The detection is frequently difficult when it is the only mechanism present because the minimum inhibitory concentration (MIC) values for carbapenems not always fall within the resistance range.1 Among the commonly recognized carbapenemase-producing Enterobacteriaceae, OXA-48-positive enterobacterial isolates have received increased attention as they are frequently associated with serious therapeutic failures in hospital settings.2
Case Report In March 2014, a 30-year-old female patient with acute lymphoblastic leukaemia treated with multi-agent chemotherapy according to HOVON ALL-100 protocol3 was transferred from the haematology ward to the Department of surgery for splenectomy. The patient had suffered necrotic hepatosplenomegaly resulting from the anticancer therapy. She had no travel history abroad. During the operation, a tumour formation including the Correspondence to: Stefana Sabtcheva, Laboratory for Clinical Microbiology, National Oncology Center – SHATO, Plovdivsko Pole Street 6, Sofia 1797, Bulgaria. Email: [email protected]
ß 2015 Edizioni Scientifiche per l’Informazione su Farmaci e Terapia DOI 10.1179/1973947815Y.0000000047
spleen, two-third of the liver and flexura lienalis was removed. Cultures made from the peritoneal exudate taken intra-operatively yielded Klebsiella pneumoniae isolate PR2899 as determined by Vitek-2 (BioMe´rieux, Marcy l’Etoile, France). Disk diffusion susceptibility tests, routinely performed in the hospital laboratory, revealed the presence of a phenotype highly suspicious for OXA-48-like enzyme. The strain had reduced susceptibility to imipenem (24 mm) and meropenem (23 mm), resistance to temocillin and penicillin/inhibitor combinations but was susceptible to extended-spectrum cephalosporins. In addition, a positive Carba NP test was observed4 and the presence of a blaOXA-48-like was confirmed by PCR. Based on these results, the treatment was carried out with intravenous combination of ceftazidime and ciprofloxacin. The infection prevention and control unit was timely notified and contact isolation precautions were maintained for the entire duration of the hospital stay. The patient was discharged home after 3 weeks of antibiotic treatment in satisfactory general condition. As a result of the immediate implementation of contact precautions, no other patients were found to be colonized or infected with the same K. pneumoniae strain. Wide range antimicrobial susceptibility testing was performed by broth microdilution and disk diffusion methods and interpreted according to CLSI
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guidelines5 except for colistin and tigecycline MICs that were interpreted using EUCAST breakpoints.6 The isolate PR2899 was resistant to all penicillins, ertapenem, tetracyclines, chloramphenicol and trimethoprim/sulphamethoxazole. It had intermediate susceptibility to tigecycline and first generation cephalosporins but remained susceptible to other carbapenems and cephalosporins, aztreonam, fluoroquinolones, aminoglycosides and colistin (Table 1). PCR screening for the presence of Ambler class A, B and D genes, followed by sequencing, identified the blaOXA-48 carbapenemase gene.7 K. pneumoniae PR2899 also possessed blaSHV-1c but no other beta-lactamase genes. The genetic environment of the blaOXA-48 gene was investigated by PCR mapping and sequencing using previously reported primers.8 These experiments revealed that the detected blaOXA-48 was flanked by two intact copies of IS1999 on truncated DTn1999.1, lacking the remnant of the tir gene usually located downstream of the blaOXA-48 and lysR. Similar truncated DTn1999.1 transposon has been recently described in OXA-48-positive Escherichia coli strain, isolated in France.9 PCR-based replicon typing designed to detect 21 replicons,10 S1-nuclease PFGE analysis11 and transfer experiments were further performed to characterize the blaOXA-48 harbouring plasmid. Repeated attempts to transfer carbapenem resistance by broth and filter mating-out assays, using a rifampin-resistant E. coli ML4909 as the recipient, failed.12 Plasmid DNA was then extracted with Nucleospin Plasmid DNA kit (Macherey-Nagel,
Germany) and transformed into chemically competent E. coli BL21(DE3) cells.13 The single transformant, obtained on Luria-Bertani selection plates with ampicillin 100 mg/L, had a resistance phenotype resembling that of the donor except for resistance to trimethoprim/sulphamethoxazole and tetracyclines (Table 1). These results are consistent with data from other studies showing that tetracycline resistance determinants were not transferred along with OXA-48-mediated carbapenem resistance.2,9 In accordance with the phenotype, the transformant was positive for the blaOXA-48 by PCR and sequencing. No other resistance genes were detected. Plasmid DNA analysis allowed the identification of the blaOXA-48 gene in a 29-kb plasmid, which was not typeable by the PCR-based replicon typing method. Multilocus sequence typing (MLST) was performed according to Protocol 2 described in K. pneumoniae MLST database (http://bigsdb.web.pa steur.fr/klebsiella/primers_used.html). K. pneumoniae PR2899 belonged to a rare ST530 with only two isolates reported so far, one strain from Poland present in the MLST database and one OXA-48 producing K. pneumoniae isolated in France.9 Unlike K. pneumoniae PR2899 described here, the French isolate harboured blaOXA-48 in the transposon Tn1999.2 on conjugative IncL/M plasmid of about 62-kb. Furthermore, PCR-based K-typing with primers for common capsular types associated with invasive disease (K1, K2, K5, K20, K54, K57) and PCR for rmpA and wcaG virulence genes was performed, as previously described.14 Although none of the seven capsular
Table 1 Antimicrobial susceptibility of OXA-48-producing Klebsiella pneumoniae PR2899, its transformant and Escherichia coli recipient strain BL21(DE3) Minimum inhibitory concentration (MIC) (mg/l)a for Antimicrobial agent
K. pneumoniae PR2899
Transformant BL21(DE3)-PR2899
E. coli BL21(DE3)
Piperacillin/tazobactam Ticarcillin/clavulanic acid Cefotaxime Ceftazidime Cefepime Aztreonam Imipenemb Meropenemb Ertapenemb Doripenem Gentamicin Tobramycin Amikacin Ciprofloxacin Levofloxacin Trimethoprim/sulfamethoxazole Colistin Polymyxin B Doxycycline Minocycline Tigecycline
w64 w128 j1 j1 j2 j2 0.75 0.38 4 0.5 j1 j1 j4 j0.25 j1 w4 1 1 w16 w16 2
w64 w128 j1 j1 j2 j2 0.75 0.125 0.25 j0.12 j1 j1 j4 j0.25 j1 j0.5 0.5 1 j2 j2 j0.25
j8 j16 j1 j1 j2 j2 0.125 0.064 0.064 j0.12 j1 j1 j4 j0.25 j1 j0.5 j0.25 0.5 j2 j2 j0.25
a MIC values were determined using SensititreH GNX2F panel (Trek Diagnostics Systems, East Grinstead, UK). b MICs of imipenem, meropenem and ertapenem were determined with LiofilchemH MIC Test Strip (MTS-LiofilchemH, Roseto degli Abruzzi, Italy).
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types was confirmed for isolate PR2899, wcaG gene was detected. This gene is involved in the synthesis of fucose, which incorporates in the capsule and interferes interaction with peritoneal macrophages as they are known to recognize bacterial surface containing mannose. The results from experimental studies of Wu et al.15 indicated that the fucose synthesis gene resided in the majority of liver abscess K. pneumoniae strains and contributed to hepatic K. pneumoniae virulence by avoiding phagocytosis, since fucose on bacteria had been implicated in immune evasion. Based on these data, the presence of wcaG gene in OXA-48 producing K. pneumoniae PR2899 could be related to the severe abdominal infectious complications developed in our patient. Interestingly, wcaG gene that was previously detected in OXA-48 producing K. pneumoniae strain from Turkey found to be non-K-typeable as well.14
Discussion Until the isolation of K. pneumoniae RP2899, carbapenem resistance in K. pneumoniae in Bulgaria was attributed to the Ambler class A and B carbapenemases.16 To our knowledge, this is the first identified Enterobacteriaceae isolate in our country possessing a carbapenem-hydrolysing oxacillinase. Previous studies on OXA-48-producing isolates indicate that plasmids carrying the blaOXA-48 gene from Enterobacteriaceae of diverse geographic origin share similar characteristics. These plasmids usually were self-transferable by conjugation, without additional resistance determinants, of IncL/M replicon type and *62 kb.2,9 We identified a smaller OXA-48-encoding plasmid of 29-kb that was not typeable by the PCR-based replicon typing method and could be transferred only by transformation. Unlike most of the reported OXA-48-positive isolates, which also co-produced an extended-spectrum beta-lactamase9 K. pneumoniae PR2899, described here, was without associated ESBL. The resulting cephalosporin-susceptible, carbapenem-non-susceptible phenotype is favourable in terms of therapeutic options but is difficult to detect in classical susceptibility testing. Our results, however, confirm that the concomitant use of temocillin and piperacillin/tazobactam disks, suggested recently by Huang et al.17 and performed routinely in our laboratory, could be a sensitive screening tool for difficult-to-detect OXA-48-positive/ESBL-negative Enterobacteriaceae. In conclusion, this is the first documented infection due to OXA-48-producing Enterobacteriaceae strain in Bulgaria. The detected enzyme has previously been found in several enterobacterial species, isolated in many countries but usually located on similar IncL/M conjugative plasmids of *62 kb. The unusual OXA-48 plasmid of 29 kb harboured by K. pneumoniae RP2899 further elucidates variability
Detection of OXA-48-producing K. pneumoniae in Bulgaria
of genetic support of blaOXA-48 carbapenemase gene. Our findings are an additional indication of the emergence of OXA-48 carbapenemase in Europe and underline the need for intensified epidemiological surveillance.
Disclaimer Statements Contributors S. Sabtcheva conceived and designed the study, interpreted the data and wrote the article. I.N. Ivanov analysed molecular data and wrote the article in part. B. Todorova collected the laboratory data and wrote the article in part. Y. Simeonov collected clinical data. E. Dobreva and K. Ivanova collected molecular data. T.Velinov and T. Kantardjiev analysed all data and revised the article. Funding No specific funding was received. Conflicts of interest The authors have no conflicts of interest to declare. Ethics approval Ethical approval was not required.
References 1 Levy Hara G, Gould I, Endimiani A, Pardo PR, Daikos G, Hsueh PR, et al. Detection, treatment, and prevention of carbapenemase-producing Enterobacteriaceae: recommendations from an international working group. J Chemother. 2013;25(3):129–40. 2 Carre¨r A, Poirel L, Yilmaz M, Akan OA, Feriha C, Cuzon G, et al. Spread of OXA-48-encoding plasmid in Turkey and beyond. Antimicrob Agents Chemother. 2010;54(3):1369–73. 3 Trial: HOVON 100 ALL. Available from: http://hovon.nl/stud ies/studies-per-ziektebeeld/all.html?action5showstudie&studie_ id569&categorie_id56. 4 Dortet L, Brechard L, Poirel L, Nordmann P. Impact of the isolation medium for detection of carbapenemase-producing Enterobacteriaceae using an updated version of the Carba NP test. J Med Microbiol. 2014;63(Pt 5):772–6. 5 Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing; twenty-fourth informational supplement. Wayne, PA: Clinical and Laboratory Standards Institute; 2014; p. M100–24. 6 European Committee on Antimicrobial Susceptibility Testing (EUCAST), Breakpoint tables for interpretation of MICs and zone diameters Version 4.0 EUCAST; 2014.Available from: http://www.eucast.org/clinical-breakpoints/. 7 Vatcheva-Dobrevska R, Mulet X, Ivanov I, Zamorano L, Dobreva E, Velinov T, et al. Molecular epidemiology and multidrug resistance mechanisms of Pseudomonas aeruginosa isolates from Bulgarian hospitals. Microb Drug Resist. 2013;19(5):355–61. 8 Giani T, Conte V, Di Pilato V, Aschbacher R, Weber C, Larcher C, et al. Escherichia coli from Italy producing OXA-48 carbapenemase encoded by a novel Tn1999 transposon derivative. Antimicrob Agents Chemother. 2012;56(4):2211–3. 9 Potron A, Poirel L, Rondinaud E, Nordmann P. Intercontinental spread of OXA-48 beta-lactamase-producing Enterobacteriaceae over a 11-year period, 2001 to 2011. Euro Surveill. 2013;18(31):20549. 10 Garcı´a-Ferna´ndez A, Fortini D, Veldman K, Mevius D, Carattoli A. Characterization of plasmids harbouring qnrS1, qnrB2 and qnrB19 genes in Salmonella. J Antimicrob Chemother. 2009;63(2):274–81. 11 Oteo J, Herna´ndez JM, Espasa M, Fleites A, Sa´ez D, Bautista V, et al. Emergence of OXA-48-producing Klebsiella pneumoniae and the novel carbapenemases OXA-244 and OXA-245 in Spain. J Antimicrob Chemother. 2013;68(2):317–21. 12 Sabtcheva S, Saga T, Kantardjiev T, Ivanova M, Ishii Y, Kaku M. Nosocomial spread of armA-mediated high-level aminoglycoside resistance in Enterobacteriaceae isolates producing
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CTX-M-3 beta-lactamase in a cancer hospital in Bulgaria. J Chemother. 2008;20(5):593–9. 13 Tu Z, He G, Li KX, Chen MJ, Chang J, Chen L, et al. An improved system for competent cell preparation and high efficiency plasmid transformation using different Escherichia coli strains. Electron J Biotechnol. 2005;8(1):113–20. 14 Turton JF, Perry C, Elgohari S, Hampton CV. PCR characterization and typing of Klebsiella pneumoniae using capsular type-specific, variable number tandem repeat and virulence gene targets. J Med Microbiol. 2010;59(5):541–7. 15 Wu JH, Wu AM, Tsai CG, Chang XY, Tsai SF, Wu TS. Contribution of fucose-containing capsules in Klebsiella
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pneumoniae to bacterial virulence in mice. Exp Biol Med. 2008;233(1):64–70. 16 Markovska R, Schneider I, Stoeva T, Bojkova K, Boyanova L, Bauernfeind A, et al. First identification of KPC-2 and VIM-1 producing Klebsiella pneumoniae in Bulgaria. Diagn Microbiol Infect Dis. 2013;77(3):252–3. 17 Huang T-D, Poirel L, Bogaerts P, Berhin C, Nordmann P, Glupczynski Y. Temocillin and piperacillin/tazobactam resistance by disc diffusion as antimicrobial surrogate markers for the detection of carbapenemase-producing Enterobacteriaceae in geographical areas with a high prevalence of OXA-48 producers. J Antimicrob Chemother. 2014;69(2):445–50.
Detection and characterization of OXA-48-producing Klebsiella pneumoniae originated in Bulgaria Stefana Sabtcheva1, Ivan N. Ivanov2, Bozhana Todorova2, Yordan Simeonov3, Elina Dobreva2, Krasimira Ivanova2, Tzvetan Velinov2, Todor Kantardjiev2 1
Laboratory for Clinical Microbiology, National Oncology Center – SHATO, Sofia, Bulgaria, 2National Reference Laboratory for Control and Monitoring of Antibiotic Resistance, Department of Microbiology, National Center of Infectious and Parasitic Diseases, Bulgaria, 3Department of Surgery, Specialized Hospital for Active Treatment in Oncology – SHATO, Sofia, Bulgaria
We report the identification of OXA-48-producing Klebsiella pneumoniae, causing peritonitis in a cancer patient admitted to the Oncology Hospital in Sofia. The isolate had reduced susceptibility to carbapenems but remained susceptible to extended-spectrum cephalosporins. PCR and sequencing confirmed the presence of blaOXA-48 gene flanked by two intact copies of IS1999 on truncated DTn1999.1. This transposon was located on unusual non-typeable 29-kb plasmid that could be transferred only by transformation. Multilocus sequence typing (MLST) indicated the presence of the sequence type ST530.This is the first documented infection due to OXA-48-producing Enterobacteriaceae strain in Bulgaria. Keywords: Carbapenem resistance, Oxacillinases, OXA-48, Klebsiella pneumoniae
Introduction Carbapenemase-mediated resistance in Enterobacteriaceae has increased in the last years, seriously compromising the management of life-threatening infections. The detection is frequently difficult when it is the only mechanism present because the minimum inhibitory concentration (MIC) values for carbapenems not always fall within the resistance range.1 Among the commonly recognized carbapenemase-producing Enterobacteriaceae, OXA-48-positive enterobacterial isolates have received increased attention as they are frequently associated with serious therapeutic failures in hospital settings.2
Case Report In March 2014, a 30-year-old female patient with acute lymphoblastic leukaemia treated with multi-agent chemotherapy according to HOVON ALL-100 protocol3 was transferred from the haematology ward to the Department of surgery for splenectomy. The patient had suffered necrotic hepatosplenomegaly resulting from the anticancer therapy. She had no travel history abroad. During the operation, a tumour formation including the Correspondence to: Stefana Sabtcheva, Laboratory for Clinical Microbiology, National Oncology Center – SHATO, Plovdivsko Pole Street 6, Sofia 1797, Bulgaria. Email: [email protected]
ß 2015 Edizioni Scientifiche per l’Informazione su Farmaci e Terapia DOI 10.1179/1973947815Y.0000000047
spleen, two-third of the liver and flexura lienalis was removed. Cultures made from the peritoneal exudate taken intra-operatively yielded Klebsiella pneumoniae isolate PR2899 as determined by Vitek-2 (BioMe´rieux, Marcy l’Etoile, France). Disk diffusion susceptibility tests, routinely performed in the hospital laboratory, revealed the presence of a phenotype highly suspicious for OXA-48-like enzyme. The strain had reduced susceptibility to imipenem (24 mm) and meropenem (23 mm), resistance to temocillin and penicillin/inhibitor combinations but was susceptible to extended-spectrum cephalosporins. In addition, a positive Carba NP test was observed4 and the presence of a blaOXA-48-like was confirmed by PCR. Based on these results, the treatment was carried out with intravenous combination of ceftazidime and ciprofloxacin. The infection prevention and control unit was timely notified and contact isolation precautions were maintained for the entire duration of the hospital stay. The patient was discharged home after 3 weeks of antibiotic treatment in satisfactory general condition. As a result of the immediate implementation of contact precautions, no other patients were found to be colonized or infected with the same K. pneumoniae strain. Wide range antimicrobial susceptibility testing was performed by broth microdilution and disk diffusion methods and interpreted according to CLSI
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guidelines5 except for colistin and tigecycline MICs that were interpreted using EUCAST breakpoints.6 The isolate PR2899 was resistant to all penicillins, ertapenem, tetracyclines, chloramphenicol and trimethoprim/sulphamethoxazole. It had intermediate susceptibility to tigecycline and first generation cephalosporins but remained susceptible to other carbapenems and cephalosporins, aztreonam, fluoroquinolones, aminoglycosides and colistin (Table 1). PCR screening for the presence of Ambler class A, B and D genes, followed by sequencing, identified the blaOXA-48 carbapenemase gene.7 K. pneumoniae PR2899 also possessed blaSHV-1c but no other beta-lactamase genes. The genetic environment of the blaOXA-48 gene was investigated by PCR mapping and sequencing using previously reported primers.8 These experiments revealed that the detected blaOXA-48 was flanked by two intact copies of IS1999 on truncated DTn1999.1, lacking the remnant of the tir gene usually located downstream of the blaOXA-48 and lysR. Similar truncated DTn1999.1 transposon has been recently described in OXA-48-positive Escherichia coli strain, isolated in France.9 PCR-based replicon typing designed to detect 21 replicons,10 S1-nuclease PFGE analysis11 and transfer experiments were further performed to characterize the blaOXA-48 harbouring plasmid. Repeated attempts to transfer carbapenem resistance by broth and filter mating-out assays, using a rifampin-resistant E. coli ML4909 as the recipient, failed.12 Plasmid DNA was then extracted with Nucleospin Plasmid DNA kit (Macherey-Nagel,
Germany) and transformed into chemically competent E. coli BL21(DE3) cells.13 The single transformant, obtained on Luria-Bertani selection plates with ampicillin 100 mg/L, had a resistance phenotype resembling that of the donor except for resistance to trimethoprim/sulphamethoxazole and tetracyclines (Table 1). These results are consistent with data from other studies showing that tetracycline resistance determinants were not transferred along with OXA-48-mediated carbapenem resistance.2,9 In accordance with the phenotype, the transformant was positive for the blaOXA-48 by PCR and sequencing. No other resistance genes were detected. Plasmid DNA analysis allowed the identification of the blaOXA-48 gene in a 29-kb plasmid, which was not typeable by the PCR-based replicon typing method. Multilocus sequence typing (MLST) was performed according to Protocol 2 described in K. pneumoniae MLST database (http://bigsdb.web.pa steur.fr/klebsiella/primers_used.html). K. pneumoniae PR2899 belonged to a rare ST530 with only two isolates reported so far, one strain from Poland present in the MLST database and one OXA-48 producing K. pneumoniae isolated in France.9 Unlike K. pneumoniae PR2899 described here, the French isolate harboured blaOXA-48 in the transposon Tn1999.2 on conjugative IncL/M plasmid of about 62-kb. Furthermore, PCR-based K-typing with primers for common capsular types associated with invasive disease (K1, K2, K5, K20, K54, K57) and PCR for rmpA and wcaG virulence genes was performed, as previously described.14 Although none of the seven capsular
Table 1 Antimicrobial susceptibility of OXA-48-producing Klebsiella pneumoniae PR2899, its transformant and Escherichia coli recipient strain BL21(DE3) Minimum inhibitory concentration (MIC) (mg/l)a for Antimicrobial agent
K. pneumoniae PR2899
Transformant BL21(DE3)-PR2899
E. coli BL21(DE3)
Piperacillin/tazobactam Ticarcillin/clavulanic acid Cefotaxime Ceftazidime Cefepime Aztreonam Imipenemb Meropenemb Ertapenemb Doripenem Gentamicin Tobramycin Amikacin Ciprofloxacin Levofloxacin Trimethoprim/sulfamethoxazole Colistin Polymyxin B Doxycycline Minocycline Tigecycline
w64 w128 j1 j1 j2 j2 0.75 0.38 4 0.5 j1 j1 j4 j0.25 j1 w4 1 1 w16 w16 2
w64 w128 j1 j1 j2 j2 0.75 0.125 0.25 j0.12 j1 j1 j4 j0.25 j1 j0.5 0.5 1 j2 j2 j0.25
j8 j16 j1 j1 j2 j2 0.125 0.064 0.064 j0.12 j1 j1 j4 j0.25 j1 j0.5 j0.25 0.5 j2 j2 j0.25
a MIC values were determined using SensititreH GNX2F panel (Trek Diagnostics Systems, East Grinstead, UK). b MICs of imipenem, meropenem and ertapenem were determined with LiofilchemH MIC Test Strip (MTS-LiofilchemH, Roseto degli Abruzzi, Italy).
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types was confirmed for isolate PR2899, wcaG gene was detected. This gene is involved in the synthesis of fucose, which incorporates in the capsule and interferes interaction with peritoneal macrophages as they are known to recognize bacterial surface containing mannose. The results from experimental studies of Wu et al.15 indicated that the fucose synthesis gene resided in the majority of liver abscess K. pneumoniae strains and contributed to hepatic K. pneumoniae virulence by avoiding phagocytosis, since fucose on bacteria had been implicated in immune evasion. Based on these data, the presence of wcaG gene in OXA-48 producing K. pneumoniae PR2899 could be related to the severe abdominal infectious complications developed in our patient. Interestingly, wcaG gene that was previously detected in OXA-48 producing K. pneumoniae strain from Turkey found to be non-K-typeable as well.14
Discussion Until the isolation of K. pneumoniae RP2899, carbapenem resistance in K. pneumoniae in Bulgaria was attributed to the Ambler class A and B carbapenemases.16 To our knowledge, this is the first identified Enterobacteriaceae isolate in our country possessing a carbapenem-hydrolysing oxacillinase. Previous studies on OXA-48-producing isolates indicate that plasmids carrying the blaOXA-48 gene from Enterobacteriaceae of diverse geographic origin share similar characteristics. These plasmids usually were self-transferable by conjugation, without additional resistance determinants, of IncL/M replicon type and *62 kb.2,9 We identified a smaller OXA-48-encoding plasmid of 29-kb that was not typeable by the PCR-based replicon typing method and could be transferred only by transformation. Unlike most of the reported OXA-48-positive isolates, which also co-produced an extended-spectrum beta-lactamase9 K. pneumoniae PR2899, described here, was without associated ESBL. The resulting cephalosporin-susceptible, carbapenem-non-susceptible phenotype is favourable in terms of therapeutic options but is difficult to detect in classical susceptibility testing. Our results, however, confirm that the concomitant use of temocillin and piperacillin/tazobactam disks, suggested recently by Huang et al.17 and performed routinely in our laboratory, could be a sensitive screening tool for difficult-to-detect OXA-48-positive/ESBL-negative Enterobacteriaceae. In conclusion, this is the first documented infection due to OXA-48-producing Enterobacteriaceae strain in Bulgaria. The detected enzyme has previously been found in several enterobacterial species, isolated in many countries but usually located on similar IncL/M conjugative plasmids of *62 kb. The unusual OXA-48 plasmid of 29 kb harboured by K. pneumoniae RP2899 further elucidates variability
Detection of OXA-48-producing K. pneumoniae in Bulgaria
of genetic support of blaOXA-48 carbapenemase gene. Our findings are an additional indication of the emergence of OXA-48 carbapenemase in Europe and underline the need for intensified epidemiological surveillance.
Disclaimer Statements Contributors S. Sabtcheva conceived and designed the study, interpreted the data and wrote the article. I.N. Ivanov analysed molecular data and wrote the article in part. B. Todorova collected the laboratory data and wrote the article in part. Y. Simeonov collected clinical data. E. Dobreva and K. Ivanova collected molecular data. T.Velinov and T. Kantardjiev analysed all data and revised the article. Funding No specific funding was received. Conflicts of interest The authors have no conflicts of interest to declare. Ethics approval Ethical approval was not required.
References 1 Levy Hara G, Gould I, Endimiani A, Pardo PR, Daikos G, Hsueh PR, et al. Detection, treatment, and prevention of carbapenemase-producing Enterobacteriaceae: recommendations from an international working group. J Chemother. 2013;25(3):129–40. 2 Carre¨r A, Poirel L, Yilmaz M, Akan OA, Feriha C, Cuzon G, et al. Spread of OXA-48-encoding plasmid in Turkey and beyond. Antimicrob Agents Chemother. 2010;54(3):1369–73. 3 Trial: HOVON 100 ALL. Available from: http://hovon.nl/stud ies/studies-per-ziektebeeld/all.html?action5showstudie&studie_ id569&categorie_id56. 4 Dortet L, Brechard L, Poirel L, Nordmann P. Impact of the isolation medium for detection of carbapenemase-producing Enterobacteriaceae using an updated version of the Carba NP test. J Med Microbiol. 2014;63(Pt 5):772–6. 5 Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing; twenty-fourth informational supplement. Wayne, PA: Clinical and Laboratory Standards Institute; 2014; p. M100–24. 6 European Committee on Antimicrobial Susceptibility Testing (EUCAST), Breakpoint tables for interpretation of MICs and zone diameters Version 4.0 EUCAST; 2014.Available from: http://www.eucast.org/clinical-breakpoints/. 7 Vatcheva-Dobrevska R, Mulet X, Ivanov I, Zamorano L, Dobreva E, Velinov T, et al. Molecular epidemiology and multidrug resistance mechanisms of Pseudomonas aeruginosa isolates from Bulgarian hospitals. Microb Drug Resist. 2013;19(5):355–61. 8 Giani T, Conte V, Di Pilato V, Aschbacher R, Weber C, Larcher C, et al. Escherichia coli from Italy producing OXA-48 carbapenemase encoded by a novel Tn1999 transposon derivative. Antimicrob Agents Chemother. 2012;56(4):2211–3. 9 Potron A, Poirel L, Rondinaud E, Nordmann P. Intercontinental spread of OXA-48 beta-lactamase-producing Enterobacteriaceae over a 11-year period, 2001 to 2011. Euro Surveill. 2013;18(31):20549. 10 Garcı´a-Ferna´ndez A, Fortini D, Veldman K, Mevius D, Carattoli A. Characterization of plasmids harbouring qnrS1, qnrB2 and qnrB19 genes in Salmonella. J Antimicrob Chemother. 2009;63(2):274–81. 11 Oteo J, Herna´ndez JM, Espasa M, Fleites A, Sa´ez D, Bautista V, et al. Emergence of OXA-48-producing Klebsiella pneumoniae and the novel carbapenemases OXA-244 and OXA-245 in Spain. J Antimicrob Chemother. 2013;68(2):317–21. 12 Sabtcheva S, Saga T, Kantardjiev T, Ivanova M, Ishii Y, Kaku M. Nosocomial spread of armA-mediated high-level aminoglycoside resistance in Enterobacteriaceae isolates producing
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Detection of OXA-48-producing K. pneumoniae in Bulgaria
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